ABSTRACT
The chromosome location, 11q21-23, is linked to loss of heterozygosity (LOH) in multiple tumors including those of breast, lung, and head and neck. To examine the process of LOH induction, the H292 cell line (human muco-epidermoid carcinoma) was irradiated or treated with anti-CD95 antibody, and individual clones isolated through two rounds of cloning. Regions of LOH were determined by screening a suite of eight polymorphic microsatellite markers covering 11p15-11q24 using fluorescent primers and genetic analyzer peak discrimination. LOH induction was observed extending through 11q21.1-11q23.3 in 6/49 of clones surviving 4 Gy and 8/50 after 8 Gy. Analysis of selected clones by Affymetrix 6.0 single nucleotide polymorphism (SNP) arrays confirmed the initial assessment indicating a consistent 27.3-27.7 Mbp deletion in multiple clones. The telomeric border of LOH mapped to a 1 Mbp region of elevated recombination. Whole genome analysis of SNP data indicated that site-restricted LOH also occurred across multiple additional genomic locations. These data indicate that 11q21.1-11q23.3, and potentially other regions of this cell line are sites of intrinsic cell-specific instability leading to LOH after irradiation. Such deletions may subsequently be propagated by genetic selection and clonal expansion.
Subject(s)
Carcinoma, Squamous Cell/genetics , Chromosomes, Human, Pair 11/genetics , Genomic Instability/radiation effects , Loss of Heterozygosity , Polymorphism, Single Nucleotide/genetics , DNA Primers/chemistry , DNA, Neoplasm/genetics , Humans , In Situ Hybridization, Fluorescence , Microsatellite Repeats , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Tumor Cells, Cultured , Tumor Stem Cell AssayABSTRACT
The location of MLL translocation breakpoints within therapy-related acute myeloid leukemia linked to drugs targeting Topoisomerase II and infant acute leukemia (IAL) are biased toward the intron 11-exon 12 region of MLL, although lacking a comprehensive explanation. To address this, blood samples were taken from breast cancer and lymphoma patients receiving Topoisomerase II inhibitor therapy. Inverse PCR analysis was used to interrogate the exon 12 region of MLL for rearrangements. Eleven of 19 observed translocations showed breakpoint junctions restricted to a single 5 bp location within exon 12. A similarly restricted distribution (11/20 breakpoint junctions) was observed in TK6 cells exposed to either estrogen (linked to IAL) or anti-CD95 antibody. The translocation hotspot was at the 5' edge of a 10-bp tract matched with a perfect palindrome, 101 bp distant. A high stringency Topoisomerase II consensus sequence binding site was noted at the geometric midpoint of the palindromes. Ligation-mediated PCR to screen TK6 cells exposed to anti-CD95 antibody showed 14/37 (38%) of DNA breaks adjacent to the 5' palindrome and 10/37 (27%) at the 3' partner. We propose a model whereby Topoisomerase II facilitates the organization of nuclease-sensitive secondary structures, stabilized by palindrome association, which are prone to rearrangement.
